Regulation of troponin and myosin conformation in thin- and thick-filament domains in the sarcomere of skeletal muscle myofibrils.

Abstract

The contractility of striated muscle is controlled by a dual-filament regulatory mechanism which couples calcium-dependent structural changes in thin filaments with those in thick filaments triggering the release of folded-helical myosin motors from the filament surface. The folded-helical conformation of motors in relaxed muscle may be stabilized in the region of the thick filament containing myosin-binding protein-C (C-zone). Moreover, in active muscle actin-bound myosin motors might locally alter the activation level of the thin filament. To test these hypotheses we developed a novel fluorescence-based approach to measure troponin and myosin orientation in subdomains of the sarcomere in isolated myofibrils. Bifunctional rhodamine probes labelling troponin-C (TnC) and the myosin regulatory light chain (RLC) were exchanged into myofibrils from rabbit psoas muscle, and the spatial distribution of probe orientations in the sarcomere was measured by fluorescence polarization microscopy. In the absence of ATP, the RLCs of actin-attached myosin motors were perpendicular to the filament axis and the orientation of the TnC probes in the region of overlap with thick filaments was markedly different from that in the rest of the sarcomere, indicating that myosin binding to actin alters the conformation of troponin in the thin filament. In relaxing conditions all TnC probes had the same conformation which was insensitive to temperature. In contrast, the RLC probes became more parallel to the filament axis as temperature was increased above 12°C, and at 30°C in the presence of 4% Dextran their orientation was more parallel in the C-zone, indicating enrichment of folded myosin motors in this filament domain. These results show directly that troponin and myosin have distinct conformations in different sarcomere subdomains in situ (supported by the Wellcome Trust and the Royal Society, UK).